Oridonin – Albiglutide chemical

Oridonin inhibits LPS-induced inﬂammation in human gingival ﬁbroblasts by activating PPARγ

Abstract

Oridonin, the major terpene isolated from Rabdosia rubescens, has been used as dietary supplement. Recently, it has been known to exhibit anti-inﬂammatory eﬀect. This study we employed an in vitro model of LPS-stimulated human gingival ﬁbroblasts to investigate the anti-inﬂammatory eﬀects and mechanism of oridonin. Oridonin (10–30 μg/mL) was administrated 1 h before LPS treatment. The results showed that oridonin signiﬁcantly inhibited inﬂammatory mediators PGE2, NO, IL-6, and IL-8 production. Immunoblotting experiments revealed that
oridonin reduced the expression of phosphorylation levels of NF-κB p65 and IκBα. Furthermore, the expression of PPARγ was up-regulated by the treatment of oridonin. Further studies showed that PPARγ inhibitor GW9662 could reverse the inhibition of oridonin on PGE2, NO, IL-6, and IL-8 production. In conclusion, oridonin inhibited LPS-induced microglia activation through activating PPARγ.

1. Introduction

Periodontal disease is a chronic inﬂammatory disease of microbial origin that often causes tooth loss in adult humans [1]. Porphyromonas gingivalis is the major bacteria that lead to periodontal disease, such as periodontitis [2]. LPS from Porphyromonas gingivalis could induce the inﬂammatory response in gingival ﬁbroblasts [3]. LPS could induce the activation of NF-κB, which subsequently leads to the release of inﬂammatory mediators [4]. These inﬂammatory mediators, including PGE2, NO, IL-6, and IL-8, result in bone resorption and inhibit bone formation [5]. Previous studies showed that increased inﬂammatory mediators were observed in patients of periodontitis [6]. Increased PGE2 production could lead to the tissue destruction and bone resorp- tion [7]. IL-6 has been known as a bone resorbing factor. A large body of evidences suggested that IL-6 plays a critical role in pathological conditions exhibiting bone loss [8]. IL-8 is a potent polymorphonuclear neutrophils chemoattractant that could induce the recruitment of neutrophils to inﬂammatory gingival sites and amply the inﬂammatory response [9]. Usually, inﬂammation is beneﬁcial to automatic defense response. However, excessive inﬂammation is also harmful and attacks the body’s own tissues. During the development of periodontitis, in- creased inﬂammatory mediators and inﬂammatory response were ob- served [10]. A large body of studies showed that inhibition of these inﬂammatory mediators had protective eﬀects against periodontitis [5,11]. These inﬂammatory mediators were regulated by the NF-κB signaling pathway [12]. A previous study showed that inhibition of NF- κB activation could inhibit the development of periodontitis [13]. Therefore, inhibition of LPS-induced inﬂammation and NF-κB activa- tion may have protective eﬀects against periodontitis. Furthermore, there is increasing evidence that many natural herbal medicines have protective eﬀects against periodontitis through inhibiting inﬂammatory response [14].

The peroXisome proliferator-activated receptor γ (PPARγ), an important transcription factor belongs to the nuclear receptor family, plays a critical role in the regulation of lipid and glucose metabolism [15]. Also, it has been known to have anti-inﬂammatory activity [16]. Activation of PPARγ had protective eﬀects against LPS-induced lung injury [17]. PPARγ ligands could inhibit LPS-induced inﬂammatory
response in rat synovial ﬁbroblasts [18]. Furthermore, PPARγ agonists could inhibit LPS-induced neuronal death in miXed cortical neurons [19]. Also, PPARγ agonist could attenuate inﬂammatory periodontal bone loss in mice [20]. PPARγ can be activated by natural fatty acids and some natural herbal compounds [21]. Studies showed many herbal compounds exhibited their anti-inﬂammatory eﬀects through activating PPARγ [22]. Previous studies showed that PPARγ agonists could inhibit LPS-induced inﬂammatory cytokine production through blocking NF- κB signaling pathway [23].

Herbal medicines have been used for the treatment of periodontal disease for a long time [24]. Rabdosia rubescens, also called ‘Dong-ling- cao’ in traditional Chinese medicine, often used to treat stomach ache, sore throat, cough, periodontitis and tumors [25,26]. Rabdosia rubescens contains a variety of active components, including diterpenoids, ﬂa- vonoids, and phenolic acids. Among these compounds, the diterpenoid compound oridonin has been known to exhibit anti-inﬂammatory ef- fects. A previous study showed that the content of oridonin could reach 7.02 mg/g using the shaking extraction method [27]. Oridonin, the major terpene isolated from Rabdosia rubescens, has been reported to have anti-oXidative, anti-tumor, and anti-inﬂammatory activities [28]. A previous study showed that oridonin induced growth inhibition and apoptosis in human gastric carcinoma cells by enhancement of p53 expression and function [29]. Oridonin has been reported to induce mitochondria-dependent apoptosis in esophageal cancer cells [30]. Oridonin has been reported to protect against LPS/D-galactosamine- induced liver injury in mice [31]. A previous study demonstrated that oridonin inhibited LPS-induced endometritis in mice through attenu-
ating TLR4/NF-κB signaling pathway [32]. Oridonin also had protective eﬀects against LPS-induced lung injury in mice [33]. Meanwhile, or-
idonin was found to inhibit LPS-induced inﬂammatory cytokines pro- duction in LPS-stimulated microglia [34]. In LPS-activated RAW264.7 macrophages, oridonin also suppressed inﬂammatory cytokines pro- duction [33]. Oridonin inhibits LPS-induced IL-6 production in hepatic stellate cells [35]. Oridonin was found to attenuate vascular inﬂammation through inhibiting MAPK and NF-κB activation [36]. In addition, oridonin was found to activating PPARγ to exhibit anticancer eﬀects [37]. However, whether oridonin could inhibit inﬂammatory response in LPS-stimulated human gingival ﬁbroblasts have not been reported. The anti-inﬂammatory mechanism of oridonin on LPS-sti- mulated human gingival ﬁbroblasts has not been clariﬁed. The present study, we detected the anti-inﬂammatory eﬀects and mechanism of oridonin on LPS-stimulated human gingival ﬁbroblasts.

2. Materials and methods

2.1. Materials

Oridonin, DMSO, GW9662, and MTT were purchased from Sigma (St. Louis, MO, USA). LPS from Porphyromonas gingivalis was obtained from InvivoGen (San Diego, CA, USA). PGE2, IL-8, and IL-6 ELISA kits were purchased from R&D systems (CA, USA). NF-κB p65, IκBα, NF-κB
p-p65, p-IκBα, and β-actin antibodies were obtained from Cell Signaling Technology (Beverly, MA). PPARγ antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Horseradish peroXidase-conjugated (HRP) secondary antibodies were purchased from Tianjin Sungene Biotech Co., Ltd. (Tianjin, China). The Griess reagent was purchased from Beyotime (Shanghai, China). All other chemicals were of reagent grade.

Fig. 2. Oridonin inhibits LPS-induced IL-6, IL-8, PGE2 and NO production in human gingival ﬁbroblasts. The data presented are the means ± SEM of three in- dependent experiments. #p < 0.05 vs. control group; *p < 0.05, **p < 0.01 vs. LPS group. Fig. 3. Eﬀects of oridonin on LPS-induced NF-κB activation. The values presented are the means ± SEM of three independent experiments. #p < 0.05 vs. control group; *p < 0.05, **p < 0.01 vs. LPS group. 2.2. Cell culture The samples of human normal gingival tissues were collected form 6 patients who were clinically free of periodontal disease. Gingival spe- cimens were taken from the non-inﬂamed periodontal tissues of 6 pa- tients who represented systemically healthy, non-smoking donors. Informed consent was obtained from all donors. Human gingival ﬁ- broblasts were isolated and cultured as described previously [38]. The experiment was in accordance with the Declaration of Helsinki and Tokyo and got the approval of the ethical committee of Harbin Medical University (approval number: 201742). 2.3. Cell viability assay MTT assay was carried to detect the cytotoXicity of oridonin on human gingival ﬁbroblasts [39]. Human gingival ﬁbroblasts were cul- tured in 96-well plates for 12 h. Then, diﬀerent concentrations of or- idonin were added 1 h before LPS treatment. 24 h later, MTT (20 μL, 5 mg/ml) was added and incubated for 4 h. Finally, the 150 μL MTT was added to each well. Optical density was detected at 570 nm using a Bio- Rad Microplate Reader. 2.4. ELISA assay 24 h after LPS treatment, the supernatants were collected. The concentration of PGE2 in the culture medium was measured by ELISA (R&D systems, USA) according to the manufacturer's instructions. The concentrations of IL-8 and IL-6 in the culture medium were tested by ELISA kits (Biolegend, USA) according to the manufacturer's instructions. For PPARγ inhibition assay, the cells were pretreated with or- idonin (30 μg/mL) for 1 h, or 10 μM GW9662 for 30 min before or- idonin incubation, and stimulated with LPS. 2.5. Nitrite determination 24 h after LPS treatment, the supernatants were collected. The level of NO in the culture medium was assessed through measuring nitrite level through Griess reagent (Beyotime, Shanghai, China) according to the manufacturer's instructions. Brieﬂy, 100 μL Griess reagent was added to 100 μL supernatant. The absorbance was detected at 520 nm. Fig. 4. Eﬀects of oridonin on PPARγ expression. The values presented are the means ± SEM of three independent experiments. #p < 0.05 vs. control group;*p < 0.05, **p < 0.01 vs. LPS group. 2.6. Western blot analysis 1 h after LPS treatment, the cells were washed three times with phosphate buﬀer saline (PBS), collected, and lysed using M-PER Mammalian Protein EXtraction Reagent (Thermo, USA) according to the manufacturer's instructions. Then, the proteins (30 μg) were loaded in each lane, separated on 12% SDS-PAGE, and electroblotted onto PVDF membranes. After blocking with 5% skimmed milk, the membranes were washed with PBST three times and each time contained 10 min. Then, the membranes were probed with primary antibodies (1:1000 dilutions in TBST): NF-κB p65, IκBα, NF-κB p-p65, p-IκBα, and β-actin at 4 °C for 24 h. The membranes were washed three times using TBST and incubated with secondary antibodies (1:2000 dilutions in TBST) at room temperature for 2 h. After wash three times with TBST, the bands were visualized using chemiluminescence reagents. The bands were quantiﬁed using Image J software (NIH, Bethesda, MD, USA). 2.7. Statistical analysis The results of this study were expressed as means ± SEM and ana- lyzed using SPSS 15.0 software (Chicago, USA). The diﬀerences be- tween groups were performed using one-way analysis of variance (one- way ANOVA) followed by Tukey's multiple comparison test. 3. Results 3.1. Eﬀects of oridonin on cell viability To detect the cytotoXicity of oridonin, human gingival ﬁbroblasts were treated with diﬀerent concentrations of oridonin in the presence or absence of LPS. As shown in Fig. 1, oridonin at the concentration of 10, 20, and 30 μg/mL did not aﬀect the cell viability of human gingival ﬁbroblasts (Fig. 1). Meanwhile, treatment of LPS did not aﬀect the viability of human gingival ﬁbroblasts. In the presence of LPS, oridonin (10, 20, 30 μg/mL) did not aﬀect the cell viability of human gingival ﬁbroblasts (Fig. 1). 3.2. Oridonin inhibits LPS-induced IL-6 and IL-8 production Cytokines IL-6 and IL-8 play critical roles in the development of periodontitis. Human gingival ﬁbroblasts were pretreated with oridonin (10, 20, 30 μg/mL) 1 h before LPS treatment. 24 h after LPS treatment, the supernatants were collected and we evaluated the eﬀects of or- idonin on LPS-induced IL-6 and IL-8 production. As shown in Fig. 2, compared with the vehicle group, LPS caused a signiﬁcant increase in IL-6 and IL-8 production. However, compared with the LPS group, LPS- induced IL-6 and IL-8 production were signiﬁcantly suppressed by pretreatment of oridonin (Fig. 2). 3.3. Oridonin inhibits LPS-induced PGE2 and NO production Human gingival ﬁbroblasts were pretreated with oridonin (10, 20, 30 μg/mL) 1 h before LPS treatment. 24 h after LPS treatment, the su- pernatants were collected and we assessed the eﬀects of oridonin on LPS-induced PGE2 and NO production. As shown in Fig. 2, compared with the vehicle group, LPS caused a signiﬁcant increase in PGE2 and NO production. However, LPS-induced PGE2 and NO production were signiﬁcantly suppressed by pretreatment of oridonin (Fig. 2). 3.4. Oridonin inhibits LPS-induced NF-κB activation To explore the mechanism of oridonin on the inhibition of in- ﬂammatory mediator production, NF-κB activation was detected. Human gingival ﬁbroblasts were pretreated with oridonin (10, 20, 30 μg/mL) 1 h before LPS treatment. 1 h after LPS treatment, the cells were collected and NF-κB activation was measured by western blot analysis. As shown in Fig. 3, compared with the vehicle group, LPS caused a signiﬁcant increase in phosphorylation levels of NF-κB p65 and IκBα. However, LPS-induced NF-κB activation was signiﬁcantly suppressed by pretreatment of oridonin (Fig. 3). 3.5. Eﬀects of oridonin on PPARγ expression To identify the upstream signaling by which oridonin mediated the inhibition of NF-κB activation, PPARγ expression was measured. Human gingival ﬁbroblasts were pretreated with oridonin (10, 20, 30 μg/mL) 1 h before LPS treatment. 1 h after LPS treatment, the cells were collected and the expression of PPARγ was measured by western blot analysis. As shown in Fig. 4, compared with the vehicle group, LPS caused a signiﬁcant decrease in PPARγ expression. However, the de- creased expression of PPARγ was signiﬁcantly increased by pretreat- ment of oridonin (Fig. 4). 3.6. PPARγ inhibitor reversed the anti-inﬂammatory eﬀects of oridonin To conﬁrm the anti-inﬂammatory mechanism of oridonin, PPARγ was inhibited by GW9662. Human gingival ﬁbroblasts were pretreated with oridonin (30 μg/mL) for 1 h, or 10 μM GW9662 for 30 min before oridonin incubation, and stimulated with LPS for 24 h. As shown in Fig. 5, the expression of PPARγ was inhibited by GW9662. And the inhibition of oridonin on inﬂammatory mediators IL-6, IL-8, PGE2, and NO production were prevented by GW9662 (Fig. 5). These results suggested oridonin exhibited its anti-inﬂammatory eﬀects through ac- tivating PPARγ. 4. Discussion In the development of periodontitis, human gingival ﬁbroblasts can be activated by bacteria and released the expression of inﬂammatory mediators, such as IL-6, IL-8, NO, and PGE2. These inﬂammatory mediators could induce the injury and destruction of periodontal tissues [40]. The present study, we aimed to investigate the anti-inﬂammatory eﬀects and mechanism of oridonin on LPS-stimulated human gingival ﬁbroblasts. In the present study, our results demonstrated that oridonin signiﬁcantly attenuated LPS-induced inﬂammatory mediator produc- tion in human gingival ﬁbroblasts. Oridonin may be used as an anti- inﬂammatory agent for the treatment of periodontitis. Periodontitis, a multifactorial microbial disease with a destructive inﬂammatory course, is characterized by destruction of periodontal support tissues. Human gingival ﬁbroblasts are the most abundant structural cell in periodontal tissue that has been known to play im- portant roles in the pathogenesis of periodontal disease [41]. Studies showed that gingival ﬁbroblasts were critical in sustaining inﬂamma- tion in periodontal disease [41]. Therefore, we chose gingival ﬁbro- blasts in this study. Previous studies showed that many bacteria could induce the development of periodontitis [42]. Among these bacteria, Porphyromonas gingivalis is the major bacterium that leads to period- ontitis [43]. Porphyromonas gingivalis was abundant in patients with periodontitis and studies demonstrated that this bacterium was asso- ciated with patient clinic-pathological characteristics [44]. LPS from Porphyromonas gingivalis has been known to have the ability to induce inﬂammation and promote tissue destruction [45]. It plays a critical role in the initiation and progression of chronic periodontitis [46]. Studies demonstrated that the elimination of pathogens and their component LPS were essential for the successful treatment of period- ontitis [47]. LPS could stimulate the cells to release the production of inﬂammatory mediators, such as IL-6, NO, and PGE2 [48]. The inﬂammatory mediators play a critical role in the pathogenesis of periodontitis [7]. Increased inﬂammatory cytokines could result in the destruction of periodontal supporting tissues [49]. Inﬂammation is a hallmark of many diseases and the continuance of this process can re- sult in tissue injury. IL-6 is a major inﬂammatory cytokine of the host response to tissue injury, infection and bone resorption [50]. NO plays a critical role in the pathogenesis of periodontal disease [51]. It stimu- lates cyclooXygenase and metalloproteinases which results in period- ontal tissue damage [51]. The production of PGE2 could induce col- lagen synthesis and regulate bone density, which subsequently lead to bone resorption [7]. Studies showed that the inhibition of inﬂammation was a beneﬁcial treatment for periodontitis [52]. Oridonin, the major terpene isolated from Rabdosia rubescens, has been reported to have anti-inﬂammatory activity [34]. Meanwhile, a large body of recent studies showed that natural herbal medicines and compounds isolated from these herbal medicines could attenuate the inﬂammatory response in LPS-stimulated human gingival ﬁbroblasts [53]. Therefore, we as- sessed the eﬀects of oridonin on inﬂammatory mediator production. In order to ﬁnd the most safe and eﬀective concentration of oridonin, MTT assay was used in this study. Our results showed that in the presence or absence of LPS, oridonin at the doses of 10, 20, and 30 μg/mL did not aﬀect the cell viability of human gingival ﬁbroblasts. Therefore, we chose the doses of 10, 20, and 30 μg/mL in this study. As shown in Fig. 2, LPS caused a signiﬁcant increase in the production of inﬂammatory mediators IL-6, IL-8, PGE2, and NO. The increases were concentration-dependent inhibited by oridonin. These results indicated that oridonin could inhibit LPS-induced inﬂammation in human gin- gival ﬁbroblasts. Previous studies showed that LPS could trigger inﬂammatory re- sponse through NF-κB signaling pathway which subsequently leads to the production of inﬂammatory cytokines [48]. These inﬂammatory mediators, such as IL-6, IL-8, PGE2, and NO were regulated by NF-κB, an important transcription factor [54,55]. Normally, NF-κB is existed as a latent form in the cytoplasm and bound to its inhibitor IκB proteins. Once stimulated by LPS, p65 is released from the IκB and translocated into the nucleus to regulate the inﬂammatory gene transcription [56]. NF-κB has been known as a target for many inﬂammatory diseases [57]. A previous study demonstrated that inhibition of NF-κB could protect mice against LPS-induced acute lung injury and kidney injury [58]. Previous studies indicated that the inhibition of NF-κB activation could protect rats against periodontitis [59]. Moreover, NF-κB is known as a target for the treatment of periodontitis [13]. To investigate the anti- inﬂammatory mechanism of oridonin, NF-κB activation was measured in this study. It has been demonstrated that NF-κB is activated in LPS- stimulated human gingival ﬁbroblasts and our results were consistent with it. As shown in Fig. 3, LPS-induced NF-κB activation was sig- niﬁcantly attenuated by the treatment of oridonin. This was consistent with a previous study showing that oridonin inhibited LPS-induced NF- κB activation in RAW264.7 cells. These results suggested that oridonin inhibited LPS-induced inﬂammatory response by inhibiting NF-κB ac- tivation in human gingival ﬁbroblasts. PPARγ is an important transcription factor belongs to the nuclear receptor family. Increasing evidence suggests that PPARγ is involved in the regulation of the inﬂammatory response [60]. Activation of PPARγ by its agonists could inhibit NF-κB signaling pathway and inﬂammatory cytokine production [61]. Recent studies demonstrated that many herbal compounds had the ability to activate PPARγ [62]. Many herbal compounds inhibited LPS-induced inﬂammatory response through ac- tivating PPARγ [22,63]. A previous study showed that the activation of PPARγ could reduce the development of periodontitis in rats [20]. Additionally, the activation of PPARγ could prevent inﬂammatory periodontal bone loss [64]. And recent studies demonstrated that asiatic acid and protocatechuic acid could inhibit LPS-induced in- ﬂammatory response in human gingival ﬁbroblasts through activating PPARγ [65,66]. In this study, our results showed that the expression of PPARγ was decreased in LPS-treated group. And this was consistent with previous studies which showed LPS could inhibit the expression of PPARγ [67,68]. Meanwhile, treatment of oridonin signiﬁcantly in- creased the expression of PPARγ. Treatment with GW9662 inhibited the expression of PPARγ. Furthermore, LPS-induced inﬂammatory med- iator production can be reversed by the treatment of PPARγ speciﬁc inhibitor GW9662. These results indicated oridonin exhibited an anti- inﬂammatory mechanism by activating PPARγ. Oridonin has been re- ported to exhibit anti-tumor activity [69]. A previous study showed oridonin targeted AE (AML1-ETO) oncoprotein [70]. And silencing AE represented the increase of expression of cell cycle-related mRNAs such as: BCL2, GATA1, TCF12, Sp1, and ERK2 (MAPK1), which activates PPARs in AML cell line Kasumi-1 [71]. In the present study, our results showed oridonin could increase the expression of PPARγ. PPARγ be- longs to the nuclear hormone receptor superfamily that can be activated by various Xenobiotics and natural fatty acids. Also, it can be activated by many natural compounds. Whether oridonin activates PPARγ through targeting AE needs to be clariﬁed in further study. In conclusion, our results demonstrated that oridonin signiﬁcantly inhibited LPS-induced inﬂammatory mediators PGE2, NO, IL-6, and IL-8 production in human gingival ﬁbroblasts. We demonstrated a novel mechanism involved in the anti-inﬂammatory activity of oridonin in LPS-stimulated human gingival ﬁbroblasts. The mechanism is through activating PPARγ, which subsequently leads to the inhibition of NF-κB activation and inﬂammatory cytokine production. Oridonin may be used as an anti-inﬂammatory agent for the treatment of periodontitis.